Substance abuse is associated with a more severe course of HIV disease as shown by the poor immune reconstitution in cART treated subjects and an increased rate of co-morbidities. The mechanisms that are involved in these poor clinical outcomes are not well understood most likely a consequence of poorly- controlled confounding factors in study cohorts, lack of quantitative assessment of drug levels, and incomplete measurement of immune function in these subjects. We have used systems biology to identify mechanisms of immune mediated protection. We have provided strong evidence that the balance between pro and anti- inflammatory pathways in cells is disrupted even in cART treated subjects. Disruption of this balance leads to poor immune reconstitution in HIV infected cART treated subjects and to increased frequencies of latently infected cells. We have access through our collaborators at FIU to a well-characterized cohort of cART treated subjects who are also cocaine users and who are well monitored for their cocaine use. Cocaine use as shown by our preliminary results account for further perturbation of the balance between pro and anti inflammatory effector molecules and cells; this results in lower levels of immune reconstitution in these subjects and potentially to higher HIV persistence. We will use state of the art approaches to address our major objective: we will delineate the molecular mechanisms that result in the immune dysfunction and HIV persistence observed in cocaine users cART treated subjects. In specific aim 1 we will use polychromatic flow cytometry to characterize the distribution and function of pro and anti-inflammatory cells including Treg subsets and determine their association to loss of T cell homeostasis and poor immune reconstitution post cART Transcriptional profiling of different memory T cell subsets will lead to the identification of molecular pathways in cocaine users which are associated to disrupted CD4 T cell homeostasis; these mechanistic findings will be validated at the single cell level by flow cytometry and by Fluidigm Biomark TM . Our preliminary results indicate that in cocaine users CD8 T cells express markers associated to deregulated homeostasis. We will determine the impact of cocaine on HIV specific CD8 T cell responses and assess if these are associated to the imbalances in the inflammatory environment or they are due to the lack of CD4 T cell help. In specific aim 2 we will assess and define mechanisms associated to the impact of cocaine on HIV persistence. We have developed two new assays that will provide critical insights to the magnitude of the HIV reservoir and to the impact of cocaine on HIV persistence. TILDA will allow us to monitor the frequencies of inducible HIV viruses and to provide a quantitative assessment of the impact of cocaine on establishment and maintenance of the HIV reservoir ex vivo in infected subjects. LARA will allow us to determine in vitro that cocaine and its metabolites can impact the establishment of HIV latency in different memory T cell subsets.